Bacillus clausii, a Foreshore-derived Probiotic ... Kim.pdf · Study protocol for induction of...

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Young Hyo Kim, MD, PhD

Associate Professor, Department of Oto-

laryngology, Inha University College of

Medicine, Incheon, Republic of Korea

Vice-Director, Inha Institute of Aerospace

Medicine, Inha University College of Medicine,

Incheon, Republic of Korea

Email: inhaorl@inha.ac.kr

Objectives: Recent studies have advocated the

use of probiotics to prevent and treat allergic

diseases. However, the immunomodulatory effects

and mechanism of probiotics in allergic airway

disease are unknown. We studied whether Bacillus

clausii (BC), a probiotic derived from mudflats, had

anti-allergic effects and compared the results with

those of Lactobacillus paracasei (LP). We also

examined whether the anti-allergic mechanisms of

probiotics are associated with hypoxia signaling.

Methods: Forty-two BALB/c mice were randomly

assigned to six experimental groups: normal

controls, ovalbumin (OVA)-induced asthmatic mice,

and OVA-induced mice orally administered with LP

or BC, at 1×109 or 5×109 CFU/ml each. We

performed differential cell count testing on

bronchoalveolar lavage fluid (BALF), lung

histopathology, serum total and OVA-specific IgE

and IgG1, titer of Th2 cytokines (IL-4, IL-5) in BALF

and pulmonary parenchyma, quantitative PCR for

heme oxygenase (HO)-1 and Hif-1α, and

immunohistochemistry for these mice.

Results: Compared to the OVA group mice, OVA-

sensitized mice treated with LP or BC showed

significantly reduced numbers of eosinophils and

neutrophils in the BALF (P<0.05). Both probiotics

also significantly reduced pulmonary inflammation

and eosinophil infiltration. Mice in the LP or BC

group had a substantially lower titer of IL-4 and IL-5

in BALF, and decreased IL-4 and IL-5 expression in

the lung parenchyma. Real-time PCR and

immunohistochemistry showed that both LP and BC

could significantly suppress HO-1 and Hif-1α

expression in asthmatic mice (P<0.05).

Conclusion: BC can attenuate murine allergic

asthma by regulating HIF-1α signaling, and its anti-

allergic effect is comparable to that of LP.

Bacillus clausii, a Foreshore-derived Probiotic, Attenuates Allergic Airway Inflammation through

Downregulation of Hypoxia SignalingHyelim Park 1,2, Ah-Yeoun Jung1,2, Chung-Soon Chang3 and Young Hyo Kim1,2

1Department of Otorhinolaryngology, Head and Neck Surgery, 2Inha Institute of Aerospace Medicine, 3Department of Biochemistry, Inha University College of Medicine, Incheon, Republic of Korea

Animals. Six-week-old female BALB/c mice

Sensitization and Challenge. For the induction of allergic asthma,

mice in the OVA group were sensitized with an intraperitoneal injection

of 20 μg OVA (Sigma-Aldrich, St. Louis, MO, USA) and 2.25 mg

aluminum hydroxide gel (alum adjuvant: Thermo, Waltham, MA, USA) in

100 μl saline on days 0 and 14. After systemic sensitization, mice were

locally challenged by intranasal instillation of 500 μg OVA on days 28,

29, and 30 (Figure 1).

Administration of probiotics.

LP1 or BC1 groups: 0.2 ml of LP or BC, at doses of 1 × 109 CFU/ml

LP5 or BC5 groups: 0.2 ml of LP or BC, at doses of 5 × 109 CFU/ml

In conclusion, this study demonstrates that BC alleviates asthmatic

symptoms in a murine model of asthma. Furthermore, we found that BC

might play a role in the HIF-1α pathway-mediated pulmonary allergic

inflammation, which probably results in immune-regulatory properties.

# Funding institution : National Research Foundation of Korea (NRF-

2018R1A6A1A03025523, NRF-2017R1C1B2001989)

Bacillus clausii (BC) is a probiotic widely used in Italy since the 1960s

for viral diarrhea in children and antibiotics-associated diarrhea. Clinical

trials have shown that BC is useful in the treatment and prevention of

recurrent respiratory infections in children.(1) This probiotic has been

demonstrated to exert immunomodulatory activities. However, few

studies have examined the anti-allergic effect of BC in individuals with

allergic diseases.

Recent studies have shown that oxidative stress can mediate a

significant effect on the pathogenesis of allergic diseases such as

asthma and rhinitis.(2) Antioxidants can attenuate asthma by suppress-

ing IgE, Th2 cytokines, mucus hypersecretion, and eosinophilic infiltra-

tion in a murine model. Furthermore, antioxidant agents are demon-

strated to decrease ROS generation and blockade of airway hyper-

responsiveness and inflammation via regulation of NF-κB and HIF-1α

levels.(3)

In this study, we demonstrated that administration of BC attenuated

allergic airway inflammation through regulation of the hypoxia signaling

pathway, whose effect is quite comparable to that of Lactobacillus

paracasei (LP) administration, one of the most well-known probiotic

strains.

INTRODUCTION

METHODS AND MATERIALS

1. Ciprandi G, Vizzaccaro A, Cirillo I, Tosca MA. Bacillus clausii exerts immuno-

modulatory activity in allergic subjects: a pilot study. Eur Ann Allergy Clin Immunol

2005;37:129–134.

2. Fatani SH. Biomarkers of oxidative stress in acute and chronic bronchial asthma. J

Asthma 2014;51:578–584.

3. Sakon S, Xue X, Takekawa M, Sasazuki T, Okazaki T, Kojima Y et al. NF-kappaB

inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and

necrotic cell death. EMBO J 2003;22:3898–3909.

CONCLUSIONS

RESULTS

REFERENCES

Figure 1. Study protocol for induction of asthma/rhinitis and oral

administration of probiotics (LP or BC)

Figure 3. After oral administration of LP or BC, the number of

eosinophils, neutrophils, and lymphocytes was considerably reduced

compared to that in the OVA group (P < 0.05, Figure 3A-E).

Infiltration of inflammatory cells and thickening of the airway epithelium

was significantly reduced after LP or BC treatment (Figure 3F). After LP

or BC treatment, the number of PAS-positive cells was significantly

decreased compared to that in the OVA group (Figure 3G).

ABSTRACT

CONTACT

Figure 2. In the LP or BC-treated group, total IgE levels tended to be decreased

compared to that in the OVA group, although without statistical significance (P >

0.05, Figure 2A). OVA-specific sIgE levels did not show a significant difference

between groups (P > 0.05, Figure 2B). On the contrary, OVA-specific IgG1 level

was significantly decreased in the LP and BC groups, compared to the OVA group

(P < 0.05, Figure 2C).

IL-4 (P < 0.001) and IL-5 (P < 0.001) were significantly increased in the OVA group,

compared to the control group. After LP or BC treatment, the titer of IL-4 and IL-5

was significantly decreased compared to that in the OVA group (P < 0.05, Figure

2D and 2E). Especially, BC1 and BC5 groups showed a statistically significant

decrease in the IL-4 titer compared to the LP group (P = 0.045).

Real-time PCR showed that IL-4 and IL-5 expression was significantly suppressed

in LP and BC groups, which is quite similar to the ELISA results (P < 0.01, Figure

2F and 2G).

Figure 4. Increased HO-1 (P < 0.001) and HIF-1α (P < 0.001) mRNA expression in the

OVA group was significantly decreased in the probiotic groups (P < 0.05, Figure 4A, and

4B). Using immunohistochemical staining, we found that HO-1 and HIF-1 α protein

expression in the LP or BC group was significantly reduced compared to that in the OVA

group.